7. Project Summary/Abstract Many of the organophosphate (OP) insecticides, such as phorate (O,O-diethyl S-ethylthiomethyl phosphorodithioate), are highly toxic with rat oral LD50's in the low mg/kg range, and they or their active metabolites are potent inhibitors of acetylcholinesterase (AChE). Phorate is consistently more toxic to females than males; for example, rat oral LD50's for males and females are 3.7 and 1.4 mg/kg, respectively. Phorate requires monooxygenase-mediated bioactivation to active anticholinesterase metabolites, similar to a number of other OP insecticides, such as parathion (O,O-diethyl O-nitrophenyl phosphorothionate). Our preliminary studies with a phorate metabolite phorate-oxon (PHO) have indicated a longer time delay and more violent signs of poisoning than with paraoxon (PXN), the active metabolite of parathion. Additionally, patterns of oxime-mediated cholinesterase reactivation differ between PHO and PXN, which is unexpected because both are diethyl phosphates, and would be expected to phosphylate cholinesterase with the same diethyl moiety and therefore display similar reactivation patterns. PHO undergoes additional bioactivation of its terminal sulfur ether to a sulfoxide, then to a sulfone. Estimates of binding energies and the unusual preliminary results observed thus far have suggested that there may be an ethoxy leaving group instead of the expected ethylthiomethyl group and that the slow bioactivation of PHO in the brain to PHO-sulfoxide and then to PHO- sulfone might be responsible for the unexpected preliminary observations. 2-PAM is the currently FDA- approved oxime AChE reactivator. However the need for a different oxime reactivator that is more effective with an unconventional phosphylating moiety as well as an oxime that can penetrate into the brain will be needed for effective phorate therapy. Our laboratories have invented and patented novel substituted phenoxyalkyl pyridinium oximes that show preliminary evidence of survival efficacy with PHO as well as convincing evidence of entry into the brain with other OP's in our rat model. Therefore the following Specific Aims are proposed: Aim 1. To confirm the leaving group of PHO or PHO metabolites through a mass spectral analysis of AChE-phosphylated peptides and through computational modeling to determine barrier height for the potential leaving groups. Aim 2. To determine bioactivation efficiency through analysis of brain and hepatic bioactivation kinetics for phorate to its several metabolites as quantified by LC/MS/MS. Aim 3. To identify more effective oxime reactivators from our novel oxime library of AChE inhibited by the three phorate metabolites through in vitro reactivation studies and limited in vivo phorate survival studies. The results of this R21 project will be the identification of a few down-selected novel oximes that can be further developed in a subsequent U01 project into effective therapeutics for phorate poisoning.